[go: up one dir, main page]

WO1998019163A1 - Dosage pour cellules en phase solide - Google Patents

Dosage pour cellules en phase solide Download PDF

Info

Publication number
WO1998019163A1
WO1998019163A1 PCT/US1997/019699 US9719699W WO9819163A1 WO 1998019163 A1 WO1998019163 A1 WO 1998019163A1 US 9719699 W US9719699 W US 9719699W WO 9819163 A1 WO9819163 A1 WO 9819163A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
analyte
solid phase
capture reagent
cells
Prior art date
Application number
PCT/US1997/019699
Other languages
English (en)
Inventor
Beverly Ann Brown
Patricia Ann Kasila
Original Assignee
Nen Life Science Products, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=24993730&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO1998019163(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Nen Life Science Products, Inc. filed Critical Nen Life Science Products, Inc.
Priority to AT97911947T priority Critical patent/ATE268904T1/de
Priority to DE69729469T priority patent/DE69729469T2/de
Priority to EP97911947A priority patent/EP0937250B1/fr
Publication of WO1998019163A1 publication Critical patent/WO1998019163A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors

Definitions

  • This invention relates to a solid phase assay and, in particular, to a solid phase cell-based assay to study cellular and biochemical processes in living cells which are responding to a stimulus by evaluating cell-related analytes without the need to attach the cell to the solid phase, without the need to radioactively label the cell either by incorporating a radioactive label into the cell or introducing a radioactive label onto the cell surface and without the need to employ cell culture techniques.
  • Cells are found in an enormous variety of sizes and shapes representing their evolutionary adaptation to different environments or to different specialized functions within a multicellular organism. Cells range in size from the smallest bacteria, only a few tenths of a micrometer in diameter, to certain marine algae and to various bird eggs with dimensions of centimeters. For all their apparent diversity, however, cells have many characteristics in common,' the most basic of which is the potential for an independent existence. That is, cells have the ability to continue living in the absence of any other cell, a capacity that requires first, a metabolic machinery capable of obtaining energy from the environment through capture of light or the degradation of chemical foodstuff, and second, the ability to use this energy to support essential life processes.
  • Cells of all types can be maintained , i.e., "cultured", in a laboratory, although not all specialized cells can be made to reproduce. Growth may take place in liquid suspension, or on the surface of agar, or as monolayers on the bottom of shallow dishes.
  • Classical cell culture technology is carried out in nutrient mixtures with cells usually cultured as a monolayer attached to a hydrophilic surface, commonly, sterile treated polystyrene.
  • the cells are usually radiolabeled either by incorporation of the radiolabel into the cell or through some type of interaction with a site on the cell surface.
  • these assays involve measurements of static systems not dynamic, i.e., living, systems which are responding to a stimulus.
  • An example of measurements involving static systems is illustrated by Czerkinsky, 1.2 Antigen-secreting Cells, Methods of Enzymatic Analysis, 3d ed., Vol.
  • WO 94/26413 published on November 24, 1994 describes an apparatus and method for studying cellular processes utilizing a vessel having a base including a layer comprising a scintillant substance and which is adapted for attachment and/or growth of cells. Cellular processes are examined by scintillation proximity assay using a reagent labeled with a radioisotope under conditions which cause a portion of the labelled reagent to become associated with or released from the cells adhering to the layer.
  • a chamber is provided containing a quenching solution, a plurality of ligand molecules and a plurality of receptor molecules.
  • the ligand molecules form a free species labelled with a beta particle emitter while the receptor is immobilized on a solid support such as the chamber wall or a microbead within the chamber.
  • Ligand introduced with the sample competes with ligand molecules already in the chamber for receptor sites on the receptor molecules and the free species is allowed to diffuse about the chamber.
  • a beta particle detector in communication with the chamber at a fixed position detects only those beta particles emitted from within the quenching distance of the quenching solution.
  • the quenching properties of the solution are used in place of the conventional separation step.
  • the process and apparatus are easily adapted for continuous monitoring of ligand level and is well suited for radioimmunoassays. There is no mention of any application with respect to a solid phase cell-based assay.
  • U.S. Patent No. 4,000,252 issued to Kosak on December 28, 1976, describes an immunoassay system which utilizes particles or other structures such as tube walls into which are embedded a fluorescer and which are coated with a ligand.
  • radiolabelled antigen and unlabelled antigen compete for binding to the ligand which is coated on the scintillant support structure.
  • Kosak discloses that upon binding of the radiolabelled antigen to the ligand, the scintillant emits light which is measured by a photomultiplier. There is no mention of any application with respect to a competitive solid phase cell-based assay.
  • U.S. Patent No. 4,568,649 issued to Bertoglio-Matte on February 4, 1986, describes ligand detection systems which employ scintillation counting.
  • the method utilizes support particles coated with ligand and impregnated with a fluorescer. When these particles are placed in an aqueous medium which contains the binding partner for the ligand in radiolabelled form, binding of the binding partner to the ligand is detected by the emission of light energy (i.e., scintillation).
  • the system is designed such that when the ligand and its binding partner are not bound to one another, the radiolabelled moiety is too far removed from the fluorescer to cause activation of the fluorescer.
  • the present invention concerns a competitive solid phase cell-based assay which comprises: a) reacting unlabeled analyte produced by at least one unlabeled cell in response to a stimulus, wherein the cell is not attached to the solid phase, with
  • this invention concerns a solid phase cell- based assay which comprises:
  • step (b) detecting and/or quantitating the amount of analyte produced by the cell from the signal generated in step (a).
  • One of the unique features of this inventions is the ability to study cellular and biochemical processes in living cells which are responding to a stimulus by evaluating cell-related analytes (i) without the need to attach the cell to the solid phase, (ii) with out the need to employ cell culture techniques and (ii) without the need to radioactively label the cell either by incorporating a radioactive label into the cell or by introducing a radioactive label onto the cell surface.
  • Unlabeled analyte produced by the unlabeled cell in response to a stimulus can be detected utilizing a competitive format in which labeled analyte competes with unlabeled analyte for binding with the capture reagent.
  • Unlabeled analyte produced by the unlabeled cell in response to a stimulus can also be detected utilizing a sandwich format in which the unlabeled analyte binds with both a capture reagent and a detector reagent.
  • the reaction of the capture reagent, analyte and detector reagent can be sequential or simultaneous.
  • the resulting capture reagent - unlabeled analyte - detector reagent complex is detected using techniques well known to those skilled in the art.
  • the advantages of the present invention include elimination of the need for a separate culturing step which is considered "heresy" by cell culture purists.
  • the cell or cells respond to stimulation without media present or the need to grow the cells in the plate. This saves at least a day or more in time and labor. Because all cells are used in the well, a smaller amount is needed. Furthermore, the cells do not need to be attached to the solid phase to be assayed. Since it is a one-well assay, the number of steps are reduced and extractions are eliminated.
  • the detection method is independent of the cell. Thus, the cell itself is not labeled.
  • the detection method can be radiometric or non-radiometric.
  • the competitive solid phase cell-based assay comprises: a) reacting unlabeled analyte produced by at least one unlabeled cell in response to a stimulus, wherein the cell is not attached to the solid phase, with
  • the invention concerns a sandwich solid phase cell-based assay which comprises:
  • step (a) reacting (i) unlabeled analyte produced by at least one unlabeled cell in response to a stimulus, wherein the cell is not attached to the solid phase, with (ii) a capture reagent specific for the analyte to be assayed, said capture reagent being immobilized on the solid phase and (iii) a detector reagent which specifically binds with the unlabeled analyte at a site different from the site where the capture reagent binds; and (b) detecting and/or quantitating the amount of analyte produced by the cell from the signal generated in step (a).
  • Living cells have the capacity to respond metabolically to environment changes in pH, chemicals, biochemicals, light, etc. Many of these responses are cascades of intracellular reactions which can include second messenger synthesis, calcium ion flux, phosphorylation or dephosphorylation events, DNA synthesis, transcription of DNA and translation of mRNA to result in protein synthesis.
  • hematopoietic colony stimulating factors are a group of glycoproteins which stimulate hemopoietic cells to proliferate, differentiate and become activated (D. Metcalf, Blood 67 (1986) 257-267).
  • Tissues or cells in culture can be shown to increase or decrease the amount of intracellular cAMP as a result of activation or deactivation of the enzyme adenylate cyclase in response to exposure to hormones or neurotransmitters (Y. Salomon, Methods ofEnzymology 195 (1991) 22-28).
  • Cell lines not normally responsive to hormones, peptides or cytokines can be made responsive by expression of the appropriate cloned receptors (C. W. Liaw, et al., Endocrinology 757 (1996) 72-77).
  • any type of cell-related analyte produced by at least one unlabeled cell in response to a stimulus can be studied using the present invention.
  • suitable analytes include, but is not limited to, receptor ligands, protein and lipid metabolite precursors, such as amino acids and fatty acids, cyclic nucleotides such as cyclic AMP, leukotrienes, cytokines, growth factors, nucleic acids and enzymes.
  • living cells are stimulated using, for example, chemical stimulation, ligand stimulation or any other means of stimulating cells known to those skilled in the art, so that analytes produced by the cell can be evaluated.
  • detection of the cell-related analyte produced in response to the stimulus is made independently of the cell.
  • Solid phase supports suitable for practicing the invention include synthetic polymer supports such as polystyrene, polypropylene, substituted polystyrene, e.g., aminated or carboxylated polystyrene; polyacrylamides; polyamides; polyvinylchloride, etc.; glass beads; agarose; nitrocellullose; nylon; polyvinylidenedifluoride; surface-modified nylon, etc.
  • the preferred support is a multiwell plate such as a microtitre plate.
  • the capture reagent and the detector reagents can be any reagents which are specific for the analyte being assayed provided that the capture reagent and the detector reagent bind to the analyte at different sites.
  • suitable capture and detector reagents include, but are not limited to, members of specific binding pairs which can be of the immune or non-immune type. Immune specific binding pairs are exemplified by antigen/antibody systems or hapten/anti- hapten systems.
  • the antibody member of the binding pair whether polyclonal or monoclonal or an immunoreactive fragment thereof, can be produced by customary methods familiar to those skilled in the art.
  • the terms immunoreactive antibody fragment or immunoreactive fragment mean fragments which contain the binding region of the antibody.
  • Such fragments may be Fab-type fragments which are defined as fragments devoid of the Fc portion, e.g., Fab, Fab' and F(ab')2 fragments, or may be so-called "half-molecule" fragments obtained by reductive cleavage of the difulfide bonds connecting the heavy chain components of the intact antibody. If the antigen member of the specific binding pair is not immunogenic, e.g., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic.
  • Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies.
  • non-immune binding pairs are biotin-avidin or biotin-streptavidin, folic acid-folate binding protein, complementary probe nucleic acids, etc.
  • the capture reagent can be immobilized directly or indirectly-, covalently or non-covalently on the solid phase using methods well known to those skilled in the art.
  • An example of direct immobilization is coating the capture reagent onto the solid phase.
  • labeled analyte which competes with unlabeled analyte for binding with the capture reagent can be labeled with a component of a reporting system or a member of a specific binding pair.
  • reporting system is discussed below.
  • any type of sandwich assay format can be used to practice the invention.
  • the reaction of the capture reagent, analyte and detector reagent can be sequential or simultaneous.
  • the detector reagent should be capable of binding unlabeled analyte at a site different from the site bound by the capture reagent. Examples of suitable detector reagents are discussed above.
  • the detector reagent or labeled analyte can be labeled directly or indirectly, covalently or noncovalently, with a component of a reporting system or a member of a specific binding pair using conventional techniques well known to those skilled in the art.
  • the term "reporting system” as used herein refers to the reporter selected and any means of linking the reporter to the detector reagent or labeled analyte or to a member of a specific binding pair. Type of specific binding pairs are discussed above.
  • the detector reagent can be linked to a reporter such as an enzyme or a radioisotope or it can be linked to a member of a specific binding pair such as biotin and the biotinylated detector reagent could then be detected by reacting it with a labeled avidin or streptavidin.
  • a reporter can be linked directly or indirectly, covalently or noncovalently, to the detector reagent, labeled analyte or member of a specific binding pair.
  • Reporters can be radioactive isotopes, enzymes, fluorogenic, colorimetric, magnetic, chemiluminescent or electrochemical materials or a first member of a specific binding pair.
  • detection of the reporter can be made directly or indirectly.
  • the detector reagent was an antibody then it could be labeled with fluorescein which can be detected directly or indirectly.
  • fluorescein In the case of direct detection, fluorescence would be detected directly.
  • fluorescein In the case of indirect detection, fluorescein can be detected using a labeled anti-fluorescein antibody. The label on the anti-fluorescein antibody would then be detected.
  • the reporter used to label analyte or the detector reagent is an enzyme, then the detector signal can be amplified by using catalyzed reporter deposition technology as described in U.S. Patent No. 5, 196, 306, the discussion of which is hereby incorporated by reference.
  • an analyte dependent enzyme activation system comprising at least one enzyme is reacted with a conjugate consisting of a detectably labeled substrate specific for the enzyme system, the conjugate reacts with the analyte dependent enzyme activation system to produce an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system wherein deposited detectable labels either directly or indirectly generate a signal which can be detected or quantitated.
  • analyte or detector reagent was labeled with the enzyme, horseradish peroxidase then after the enzyme-labeled analyte bound with the capture reagent, it could then be reacted with a conjugate such as biotin tyramine.
  • the conjugate would be activated and the activated biotin tyramine would deposit wherever there was receptor for the activated conjugate.
  • the deposited biotin could then be detected through reaction with a labeled streptavidin or labeled avidin.
  • EXAMPLE 1 Adenylate Cyclase FlashPlate® Assay Cells (HEK293 (ATCC) with a cloned CRF2 receptor) were harvested from tissue culture flasks using Versene EDTA (GIBCO). The cells were pelleted by centrifuging for 5 minutes at 1000 xg at room temperature. The pellet was resuspended in stimulation buffer (IX Phosphate Buffered Saline (PBS), 0.1% Bovine Serum Albumin (BSA), 500 ⁇ M 3-Isobutyl-l-methylxanthine (IBMX), 0.09% 2-chloroacetamide), washed by centrifugation for 5 minutes at 1000 xg at room temperature.
  • stimulation buffer IX Phosphate Buffered Saline (PBS), 0.1% Bovine Serum Albumin (BSA), 500 ⁇ M 3-Isobutyl-l-methylxanthine (IBMX), 0.09% 2-chloroacetamide
  • the pellet was resuspended in stimulation buffer to a concentration of 1 x 10 ⁇ cells/mL. Fifty microliters of cells were then added to a FlashPlate® (DuPont) well that had been previously coated with anti-cAMP antibody (DuPont) (Sheep anti- rabbit antibody (DuPont) was coated overnight, blocked with 1% BSA then coated with rabbit anti-cAMP antibody). A set of cAMP standards were prepared in the stimulated buffer used for the cells to concentrations of 0, 10, 25, 50, 100, 250, 500, and 1000 pmol/mL by diluting down to the appropriate concentration from a stock standard of 5000 pmol/mL (DuPont). Fifty microliters of standards were added to the FlashPlate® wells.
  • the cells were then stimulated with a chemical agonist (Forskolin) (Sigma) at concentrations of 0, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 ⁇ M or a ligand to a receptor on the cell membrane (Sauvagine is a ligand for the corticotropin releasing factor 2 receptor) (Penninsula Labs) at concentration of 0, 12.5, 25, 50, 100, 200, 400, and 800 nM which then activates adenylate cyclase to produce cAMP.
  • a chemical agonist forskolin
  • Tauvagine is a ligand for the corticotropin releasing factor 2 receptor
  • the detection mix 13.6% Acetic acid, sodium salt trihydrate, 0.6% EDTA disodium salt, 0.09% sodium azide, 10 mM calcium chloride, and 0.35% Triton X-100 containing an ⁇ 5j labeled cAMP tracer (DuPont) was added to the well.
  • the cAMP that was produced by the stimulated cells then competed with the 125j_ labeled cAMP for binding to the FlashPlate® through the anti-cAMP antibody.
  • the FlashPlate® was read for counts per minute bound to each well on a TopCountTM scintillation counter (Packard). The sample values were interpolated from the stand curve.
  • Table 1 shows the Forskolin stimulation and Table 2 shows the ligand stimulation.
  • the negative control for both assays was the cells not stimulated.
  • the assay showed a significant increase in cAMP produced and detected for the stimulated cells and not for unstimulated cells.
  • Leukotriene C4 FlashPlate® Assay Whole blood (3 mL) from a human donor was collected in a heparinized syringe.
  • the leukocytes were separated from the whole blood by lysing 0.1 mL whole blood with 1.0 mL of a red blood cell lysing reagent (0.168M NH4CI, 0.01M KHCO3, 0.1 mM EDTA). After 10 minutes at room temperature the leukocytes were pelleted by high speed centrifugation (10 seconds at 14,000 xg). The pellet was resuspended in PBS and washed by centrifugation. The pellet was resuspended in PBS containing calcium chloride and magnesium chloride.
  • LTC4 standards were prepared in the PBS to concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 ng/0.1 mL.
  • the cells and the standards were added to the FlashPlate® (DuPont) that had been previously coated with sheep anti-rabbit antibody (DuPont), blocked, and then coated with rabbit anti- LTC4 antibody (DuPont).
  • the cells were either stimulated with calcium ionophore A23187 (10 ⁇ L of a 2 ⁇ M stock) (Sigma) to produce LTC4 or as a negative control, water (10 ⁇ L) was added to the cells.
  • Ionophor A23187 stimulated cells produced more LTC4 than the unstimulated cells and that the LTC4 was able to be detected by this method.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Fuel Cell (AREA)
  • Developing Agents For Electrophotography (AREA)
  • Silicon Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cette invention se rapporte à un dosage pour cellules en phase solide, qui peut être utilisé pour étudier les processus cellulaires et biochimiques dans des cellules vivantes qui répondent à un stimulus, en évaluant les analytes correspondants aux cellules, sans qu'il soit nécessaire de fixer la cellule à la phase solide, sans qu'il soit nécessaire de marquer radioactivement la cellule soit en incorporant dans la cellule une étiquette radioactive soit en introduisant une étiquette radioactive sur la surface de la cellule, et sans qu'il soit nécessaire d'employer des techniques de culture cellulaire.
PCT/US1997/019699 1996-10-29 1997-10-29 Dosage pour cellules en phase solide WO1998019163A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AT97911947T ATE268904T1 (de) 1996-10-29 1997-10-29 Zellbasiertes festphasentest
DE69729469T DE69729469T2 (de) 1996-10-29 1997-10-29 Zellbasierter festphasenassay
EP97911947A EP0937250B1 (fr) 1996-10-29 1997-10-29 Dosage pour cellules en phase solide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/744,718 1996-10-29
US08/744,718 US5739001A (en) 1996-10-29 1996-10-29 Solid phase cell-based assay

Publications (1)

Publication Number Publication Date
WO1998019163A1 true WO1998019163A1 (fr) 1998-05-07

Family

ID=24993730

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/019699 WO1998019163A1 (fr) 1996-10-29 1997-10-29 Dosage pour cellules en phase solide

Country Status (5)

Country Link
US (1) US5739001A (fr)
EP (1) EP0937250B1 (fr)
AT (1) ATE268904T1 (fr)
DE (1) DE69729469T2 (fr)
WO (1) WO1998019163A1 (fr)

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0863402B1 (fr) 1997-03-03 2005-09-14 Amersham Biosciences UK Limited In-situ d'extraction de cellules et procédé
US7632651B2 (en) * 1997-09-15 2009-12-15 Mds Analytical Technologies (Us) Inc. Molecular modification assays
US7745142B2 (en) * 1997-09-15 2010-06-29 Molecular Devices Corporation Molecular modification assays
US20050227294A1 (en) * 1997-09-15 2005-10-13 Molecular Devices Corporation Molecular modification assays involving lipids
US7070921B2 (en) 2000-04-28 2006-07-04 Molecular Devices Corporation Molecular modification assays
US20050089918A1 (en) * 1998-02-23 2005-04-28 Amersham Biosciences Uk Limited In-situ cell extraction and assay method
JP2002544488A (ja) * 1999-05-07 2002-12-24 クアンタム ドット コーポレイション 半導体ナノクリスタルを用いて分析物を検出する方法
US6686171B2 (en) * 1999-05-10 2004-02-03 Tropix, Inc. Competitive chemiluminescent assay for cyclic nucleotide monophosphates
US6794156B2 (en) * 1999-05-10 2004-09-21 Applera Corporation Cell growth, induction and lysis in an antibody-coated microplate for use in an ELISA
US20030157586A1 (en) * 2002-02-21 2003-08-21 Martin Bonde Device and method for conducting cellular assays using multiple fluid flow
US7771999B2 (en) * 2004-04-28 2010-08-10 Vaxdesign Corp. Disease model incorporation into an artificial immune system (AIS)
US8071373B2 (en) 2004-04-28 2011-12-06 Sanofi Pasteur Vaxdesign Corp. Co-culture lymphoid tissue equivalent (LTE) for an artificial immune system (AIS)
US20070141552A1 (en) * 2004-04-28 2007-06-21 Warren William L Automatable artificial immune system (AIS)
US7785806B2 (en) * 2004-04-28 2010-08-31 Vaxdesign Corporation Method for determining the immunogenicity of an antigen
US20060275270A1 (en) * 2004-04-28 2006-12-07 Warren William L In vitro mucosal tissue equivalent
US8298824B2 (en) 2004-04-28 2012-10-30 Sanofi Pasteur Vaxdesign Corporation Methods of evaluating a test agent in a diseased cell model
US7709256B2 (en) * 2004-04-28 2010-05-04 Vaxdesign Corp. Disease model incorporation into an artificial immune system (AIS)
US8030070B2 (en) * 2004-04-28 2011-10-04 Sanofi Pasteur Vaxdesign Corp. Artificial lymphoid tissue equivalent
US7785883B2 (en) * 2004-04-28 2010-08-31 Vax Design Corp. Automatable artificial immune system (AIS)
US7855074B2 (en) 2004-04-28 2010-12-21 Vaxdesign Corp. Artificial immune system: methods for making and use
US20070134685A1 (en) * 2005-09-06 2007-06-14 Invitrogen Corporation Control of chemical modification
EP1969366B1 (fr) 2005-12-21 2015-10-21 Sanofi Pasteur VaxDesign Corporation Centres germinaux in vitro
EP1963491B1 (fr) * 2005-12-21 2011-01-12 VaxDesign Corporation Methode in vitro pour evaluer la reaction potentielle d'un animal a un agent
ATE525093T1 (de) * 2006-06-27 2011-10-15 Sanofi Pasteur Vaxdesign Corp Modelle für die bewertung von impfstoffen
WO2009048661A1 (fr) 2007-07-16 2009-04-16 Vaxdesign Corporation Constructions tissulaires artificielles comprenant des cellules alvéolaires et leurs procédés d'utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6111663A (ja) * 1984-06-28 1986-01-20 Ono Pharmaceut Co Ltd 酵素免疫測定法に用いられる酵素で標識されたロイコトリエン類
WO1993002361A1 (fr) * 1991-07-23 1993-02-04 Medgenix Diagnostic S.A. Dosage in vitro de molecules au niveau de leur site de production par des cellules en culture
WO1993007493A1 (fr) * 1991-10-01 1993-04-15 Alain Ladislas De Weck Procede de detection des sulfidoleukotrienes dans les tissus et les fluides biologiques et son application au diagnostic d'allergies et d'autres maladies inflammatoires

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4000252A (en) * 1974-01-04 1976-12-28 Kenneth Kosak Immunoscintillation cell
ES525541A0 (es) * 1982-10-12 1984-12-16 Dynatech Lab Un recipiente que tiene al menos una cavidad o pocillo para contener al menos una muestra de ensayo durante una medicion fluometrica.
US4568649A (en) * 1983-02-22 1986-02-04 Immunex Corporation Immediate ligand detection assay
US4626513A (en) * 1983-11-10 1986-12-02 Massachusetts General Hospital Method and apparatus for ligand detection
FR2615622B1 (fr) * 1987-05-19 1994-05-06 Ire Medgenix Sa Dosage plasmatique de monokines
SE8803602D0 (sv) * 1988-10-11 1988-10-11 Wallac Oy A sample plate with a plurality of sample wells or vials intended for radiolabeled binding assays
SE8804657D0 (sv) * 1988-12-27 1988-12-27 Gerald Potter Colin Support structure for use in a proximity assey
DE69407923T2 (de) * 1993-05-17 1998-04-30 Amersham Int Plc Vorrichtung und verfahren zum nachweis zellularer und biochemischer prozesse

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6111663A (ja) * 1984-06-28 1986-01-20 Ono Pharmaceut Co Ltd 酵素免疫測定法に用いられる酵素で標識されたロイコトリエン類
WO1993002361A1 (fr) * 1991-07-23 1993-02-04 Medgenix Diagnostic S.A. Dosage in vitro de molecules au niveau de leur site de production par des cellules en culture
WO1993007493A1 (fr) * 1991-10-01 1993-04-15 Alain Ladislas De Weck Procede de detection des sulfidoleukotrienes dans les tissus et les fluides biologiques et son application au diagnostic d'allergies et d'autres maladies inflammatoires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 010, no. 157 (P - 464) 6 June 1986 (1986-06-06) *

Also Published As

Publication number Publication date
EP0937250B1 (fr) 2004-06-09
DE69729469D1 (de) 2004-07-15
US5739001A (en) 1998-04-14
EP0937250A1 (fr) 1999-08-25
ATE268904T1 (de) 2004-06-15
DE69729469T2 (de) 2005-06-09

Similar Documents

Publication Publication Date Title
EP0937250B1 (fr) Dosage pour cellules en phase solide
AU695012B2 (en) Immunoassay for determination of cells
AU645014B2 (en) Methods, reagents and test kits for determination of subpopulations of biological entities
US5821066A (en) Simple, rapid method for the detection, identification and enumeration of specific viable microorganisms
EP0559738B1 (fr) Procedes servant a detecter et a quantifier la presence de sous-ensembles cellulaires dans des sous-populations d'une population de cellules melangees
US6806056B2 (en) Fluorescent capture assay for kinase activity employing an anti-phosphotyrosine antibody as both the capture agent and detecting agent
EP0239318B1 (fr) Dosage avec emploi de membres d'une paire liante sur les particules et sur un filtre ou une membrane
WO1989010566A1 (fr) Procede pour former et pour utiliser des microgouttes
JPH09509248A (ja) 検定方法
CA2179823C (fr) Methode de titrage mettant en oeuvre deux types de particules distinguables
Kricka Chemiluminescence and bioluminescence
US6054282A (en) Method for detecting biological interactions especially in receptor binding assays
CN104677889B (zh) 一种基于鲁米诺功能化的磁性免疫探针检测甲胎蛋白的方法
CA2030175A1 (fr) Methodes pour le diagnostic et la surveillance des affections rhumatismales
JP3340715B2 (ja) 被検体、その相互作用又は反応動力学を定量的又は定性的に測定する方法及びサンプルキャリア
US4544629A (en) Receptor-based histamine assay
WO1989010786A2 (fr) Procede de formation et d'utilisation de microgouttelettes
CA1231047A (fr) Epreuve de diagnostic des affections rhumatologiques
JPS6247555A (ja) シンチレ−シヨン近接定量法
US6423549B1 (en) Phycoerythrin labeled thyronine analogues and assays using labeled analogues
WO2001034788A1 (fr) Dosage in vitro destine a l'estimation de la cytotoxicite d'un agent candidat
EP0589612A2 (fr) Essai de liaison de piégeage de label, non-séparation

Legal Events

Date Code Title Description
AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1997911947

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997911947

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1997911947

Country of ref document: EP